The present invention relates to methods of isolating hemocyanins from hemolymph of three Chilean species of limpet. Specifically, the present invention relates to hemocyanins obtained from hemolymph of Fissurella latimarginata, Fissurella cumingi and Fissurella maxima. 
Hemocyanins are proteins whose function is oxygen transport in several mollusk and arthropod species. These proteins contain copper, which confers the characteristic blue colour after binding with oxygen. Hemocyanins of mollusks and arthropods are useful in various applications in immunology, immunochemistry and biotechnology, because they are effective immunogens inducing the synthesis of large amounts of specific antibodies and the activation of T-lymphocytes. Some of the applications of hemocyanins include: 1) their use as experimental antigens in studies of the immune response of vertebrates, 2) their use as carrier proteins used in the production of monoclonal and polyclonal antibodies against different substances, which by themselves are not immunogenic (haptens) such as synthetic peptides, recombinant proteins of microorganisms, plants, animals and humans, toxins, medications, hormones, drugs and chemicals (antibodies generated can be used in the production of kits for diagnosis, organic molecule screening and therapies for animals and humans diseases), 3) their use as a non specific immunostimulanting agent in the therapy of certain types of cancer, and 4) their use as a diagnosis reagent for diseases caused by parasites, such as Schistosomiasis.
The most frequent use of hemocyanins, particularly hemocyanin from the keyhole limpet or KLH (Megathura crenulata), is in biomedicine and biotechnology, as a carrier protein to develop antibodies against peptides and haptens of diagnostic interest, such as toxins, hormones, drugs and chemicals from different origin. The main clinical use of KLH is as a non-specific immunostimulanting agent in the treatment of surface bladder carcinoma.
Hemocyanin present in hemolymph of some mollusks has a high immunogenic capability in vertebrates due to its high molecular weight (from 4.5×106 to 1.4×107), its phylogenetic origin far from vertebrates and also, because it is a glycoprotein.
The basic structure of hemocyanins is made up by subunits arranged forming a decamer (see FIG. 1). In gastropods, decamers are normally associated, forming di-decamers, conferring on them a D5 simmetry, which can be considered similar to that of viruses. This molecule contains a large amount of ε-amine groups of lysins, which allow their conjugations with other proteins and haptens. Conjugation is performed through traditional methods based on carbodiimide or glutaraldehyde or hydroxysuccinimide esters. A hemocyanin molecule commonly accepts up to about 100 hapten molecules without lossing its immunogenicity.
W. O. Weigle (“Immunochemical Properties of Hemocyanin”, Immunochemistry. vol. 1, pp. 295-302, 1964) teaches the immunochemical properties of hemocyanin obtained from Megathura crenulata, but also demonstrates that the preparation used contained at least two antigenic components, according to results obtained by gel difussion, immunoelectrophoresis and cellulose acetate electrophoresis.
J. E. Mellema and A. Klug (“Quaternary Structure of Gastropod Hemocyanin”. Nature vol. 239, pp. 146-150, 1972) demonstrated the presence of a quaternary structure in hemocyanins obtained from three different gastropods (Kelletia kelletia, Busycon canaliculatum and Helix pomatia). For all of them, hemocyanins form cylindrical particles, with no basic structual differences found. Variations seem to reflect differences due to preparations methods.
H. B. Herscowitz et al. (“Immunochemical and Immunogenic Properties of Purified Keyhole Limpet Hemocyanin”, Immunology. vol. 22, pp. 51-61, 1972) described a method of obtaining a relatively homogeneous preparation of Megathura crenulata's hemocyanin. The procedure described by Herscowitz et al. included the use of ion-exchange cromatography in DEAE-cellulose, followed by agarose-bead gel filtration. The product obtained was analized through agar immunoelectrophoresis, polyacrilamide gel electrophoresis and agar double diffusion, showing that the purified preparation contained just one primary antigenic component, but the raw material contained multiple antigenic components.
J. Markl et al. (“The role of two distinct subunit types in the architecture of keyhole limpet hemocyanin (KLH), Naturwissenschaften. vol 78, pp. 512-514, 1991), through transmission electronic microscopy with negative staining, ultracentrifugation, dissociation in adequate buffers and the subsequent native polyacrilamide gel chromatography, demonstrated that hemocyanin from Megathura crenulata contains two types of molecules: one made up of 8 functional units named type-1 and the other made up by 7 functional units, named type-2.
J. R. Harris et al. (“Immunoelectron microscopy of hemocyanin from the Keyhole Limpet (Megathura crenulata): A parallel subunit model”, Journal of Structural Biology. vol 111, pp. 96-104, 1993) used anti-hemocyanin monoclonal antibodies from Megathura crenulata in transmission electronic microscopy with negative staining and found that, within each decamer, exist a parallel array of subunits.
R. D. Swerdlow et al. (“Keyhole limpet hemocyanin: structural and functional characterization of two different subunits and multimers”, Comparative Biochemistry and Physiology. vol 113B, pp. 537-548, 1996) demonstrated through immunoelectrophoresis analysis that both molecular forms described for hemocyanin (KLH1 and KLH2) do not include common epitopes and differ with regard to the immune response induced in experimental animals.
S. M. Sóhngen et al. (“Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH)”, European Journal of Biochemistry. vol 248, pp. 602-614, 1997) studied the structure of KLH1 and KLH2 through analytical scanning electronic microscopy, polyacrilamide gel electrophoresis, immunoelectrophoresis, controlled proteolytic digestion and amino acid sequencing. They found that these functional subunits differ both in size and in the preferential aggregation form. KLH1 and KLH2 were found to have a molecular mass of 400 KDa and 345 KDa, respectively. The subunit of KLH1 was found to have 8 different functional domains of 45 to 65 Da. In contrast, subunit KLH2 was found to have 7 functional domains and to lack the C-terminal domain named 1h, present in KLH1. In addition, KLH subunits differed with regard to the association and dissociation rates.
C. A. Olsson et al. (“Immunologic reduction of bladder cancer recurrent rate”, Journal of Urology Vol. 111, pp 173-176, 1974) looked at a non-specific immunostimulation with Keyhole limpet hemocyanin in 29 patients (26 men and 3 women, between 30 to 93 year range, naive for radiotherapy or chemotherapy) diagnosed with transitional surface bladder carcinoma. Specifically, patients were divided in two groups, according to the disease background. Group 1, included 10 patients with 13 bladder tumor episodes receiving hemocyanin 5 mg subcutaneously at the study initiation. Group 1 was considered the control group, because the 2-year tumor frequency before the treatment is known. Group 2 included 19 newly diagnosed patients (1 year), who were treated through transurethral resection only. 9 patients in Group 2 were immunized with hemocyanin and 10 were not, comprising the control group. A significant reduction of frequency in tumor recurrence in a 2-year follow-up period was found in both of hemocyanin-treated patients.
C. D. Jurincic et al. (“Immunotherapy in bladder cancer with Keyhole-limpet hemocyanin: A randomized study”, Journal of Urology. vol 139, pp 723-726, 1988) presented results from two studies aimed to assess the immunotherapeutic effect of the Keyhole limpet hemocyanin in patients diagnosed with surface bladder cancer. The first study began in 1982 and involved 44 patients undergoing recurrent surface bladder cancer surgery. Previous to therapy through vesical instillation with hemocyanin, patients were immunized with hemocyanin 1 mg intracutaneously and received 10 mg monthly by vesical instillation. The control group was given mitomycin C 20 mg monthly. Out of 21 patients treated with hemocyanin, 20 (95.2%) showed partial and complete prevention of the tumor and 3 (14.2%) showed tumor recurrence, compared with 9 (39.1%) of the control group. The second study began in 1984 with 81 patients given the same treatment as the previous study. No control group was present in the follow up study. 17 Patients (20.9%) were found with tumor recurrence and 70 patients (86.4%) showed partial and full prevention. In patients treated with Keyhole limpet hemocyanin, no local or systemic adverse effects were found. These studies have been complemented with C. D. Jurincic et al. (Effect of keyhole limpet hemocyanin (KH) and bacillus Calmette-Guerin (BCG) instillation on carcinoma in situ of the urinary bladder. Anticancer Res. 1995;15:2771-2776) and C. D. Jurincic et al. (Keyhole limpet hemocyanin for carcinoma in situ of the bladder: a long-term follow-up study. Eur. Urol. 2000;37 Suppl 3:45-9), who have provided further evidence regarding immunotherapy of surface bladder cancer using keyhole limpet hemocyanin.
J. Flamm et al. (“Recurrent superficial transitional carcinoma of the bladder: Adjuvant chemoherapy versus immunotherapy. A prospective randomized trial”, Journal of Urology, vol 144, pp.260-263,1990) presented results of a comparative study on prevention and treatment of the transitional bladder cancer standard therapy with etoglucids versus immunotherapy with Keyhole-limpet hemocyanin in 84 patients with high-risk tumor recurrence. Before the instillation started, all patients were subjected to a tumor transurethral removal and, therefore, were tumor-free at the time of treatment initiation. The group of patients treated with etoglucids received 0.565 mg weekly over 6 weeks and then monthly for 1 year. The group of patients treated with hemocyanin was immunized with 1 mg intracutaneously and then received vesical instillations 30 mg each over 6 weeks and then monthly for 1 year. The recurrence rate was 60.9% in patients treated with etoglucid versus 55.3% in patients treated with hemocyanin. The difference between both treatments was not significant, concluding that immunotherapy of this kind of recurrent tumors with hemocyanin is comparable in efficacy to the standard treatment.
D. L. Lamm et al. (“Immunotherapy of murine bladder cancer with Keyhole Limpet hemocyanin (KLH)” Journal of Urology. vol. 149, pp. 648-652, 1993) provided results from immunotherapy with Keyhole limpet hemocyanins in a bladder cancer experimental model in mice of the C3H/HeN strain implanted with MBT2 cells, demonstrating that hemocyanin is an immunomodulator with significant antitumoral activity in this animal model. Recently, D. L. Lamm et al. (Keyhole limpet hemocyanin immunotherapy of bladder cancer: laboratory and clinical studies. Eur Urol. 2000;37 Suppl 3:4144)developed immunotherapy with Keyhole-limpet hemocyanin in humans through laboratory and clinical studies.
M. M. Wishahi et al. (Keyhole limpet hemocyanin immunotherapy of bladder cancer: A new treatment modality? phase 11 trial: Superficial bladder cancer”, Journal of Urology. Vol 153, pp. 926-928, 1995) provided results of treatment with Keyhole limpet hemocyanin of 13 patients presenting with transitional bladder tumors associated with urinary schistosomiasis. Immunotherapy with hemocyanin was found to reduce tumoral recurrence rate to 15.4% compared to 76.9% before therapy.
It was also demonstrated that Keyhole limpet hemocyanin has both adjuvant and immunostimulant properties. As such, hemocyanin compounds are referred to as immunomodulators. J. Banchereau el al. (Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine. Cancer Res. 2001;61:64518) and P Hersey et al. (Phase 1/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma. Cancer Immunol. Immunother. 2004;53:125-34) demonstrated the use of Keyhole limpet hemocyanin as immunomodulator in the treatment of disseminated melanoma. In particular, the group applied hemocyanin as immunomodulator for therapy against melanoma through dendritic cells loaded with a tumor extract.
Based on the state of the art, traditionally in all the above mentioned studies, the hemocyanin drawn from the Keyhole Limpet has been used, which corresponds to the mollusk Megathura crenulata, whose haemocyanyn is known as KLH.
Another form of hemocyanin includes hemocyanin obtained from the Chilean mollusk “Loco” (Concholepas concholepas), named CCH (De loannes & cols., (2004). Hemocyanin of the mollusk Concholepas concholepas exhibits an unusual heterodecameric array of subunits (J. Biol. Chem., 279, 26134-42. ) and shows immunostimulant properties similar to KLH, used for generation of monoclonal and policlonal antibodies. In particular, it was shown previously that hemocyanin from Concholepas concholepas has two subunits exhibiting different behaviors, showing that the subunit CCH-A is more immunogenic than subunit CCH-B (De loannes & Becker, 2005, US Patent 2005/0020486 A1).
Over-exploitation of Keyhole Limpet has created a KLH shortage in the international market, and the encouraging results of immunostimulation and immunotherapy of bladder cancer in humans, have driven the search for molecules with similar characteristics.
From the above stated, the need for alternative substances to replace or complement the use of KLH, complying with proper characteristics related to the immune response, becomes evident.